Process for the separation of solid materials from microorganisms

ABSTRACT

Solid materials are separated from micro organisms by a process which comprises growing them at a fermentation temperature, conditioning them by cooling them without freezing and then heating to above the fermentation temperature without permitting substantial recovery from the conditioning stage.

THIS INVENTION relates to a process for the separation of solidmaterials from microorganisms.

It may be advantageous to separate protein from microorganisms eitherbecause the protein itself is required or because other components ofthe microorganisms are required to be separated from protein.

An example of the latter case is in the production of plastics materialsusing microorganisms. Polyhydroxyalkanoic acids (PHA) are usefulplastics materials which can be produced by this means and it isnecessary if they are to be produced in an acceptable state of purity tofree them from other cellular material which includes proteins.

It is known to break down cells containing PHA by heat and then tosolubilise and preferably to decompose proteinaceous material forexample by the use of enzymes. However, there is a tendency for proteinsto denature during such treatment and this may make them resistant tothe action of solubilising and decomposing agents for example enzymes;they are then difficult to remove from the PHA.

The PHA is preferably a thermoplastic hydroxybutyrate polyester, whichmay be a copolymer comprising other hydroxyalkanoate units for examplehydroxyvalerate units. Usually it will be of D(-) configuration andsuitably comprises up to 30% and preferably 3 to 25% hydroxyvalerateunits, the balance being preferably substantially all hydroxybutyrateunits, these percentages being by weight. Its molecular weight issuitably at least 50,000 and preferably at least 100,000 for example atleast 500,000.

We have now found that if microorganisms are grown in a fermentationstage at a fermentation temperature at which they multiply rapidly andare then conditioned to render them susceptible to thermal shock in aconditioning stage by cooling them substantially below the fermentationtemperature, to a conditioning temperature which is preferably by atleast 10° C. and more preferably by at least 20° C. below thefermentation temperature without any substantial freezing, preferablymaintained at the conditioning temperature for at least 1 hour andpreferably at least 3 and more preferably at least 4 hours for example 4to 12 hours, and then subjected to a protein separation stage by heatingthem to a protein separation temperature 10° to 150° C. and preferably15° to 120° C. for example 20° to 65° C. above the fermentationtemperature without permitting any substantial recovery from theconditioning effect, that the separation of protein is improved comparedwith that obtained if no conditioning stage is provided. Thefermentation, cooling and heating stages are preferably carried out inthe presence of water. Thus the severity of the heat treatment and thusof the denaturing of the proteins may be less for a similar level ofprotein removal and/or more protein may be removed. The separatedprotein may be removed from the remaining cellular material immediatelyor after further processing. If desired protein removal may be carriedout in the presence of a surfactant.

Thus the invention comprises a process in which a solid component isrecovered from a microorganism which comprises growing the microorganismat a fermentation temperature, conditioning it to render it susceptibleto thermal shock in a conditioning stage by cooling it substantiallybelow the fermentation temperature without any substantial freezing,solubilising protein contained in the microorganism by heating themicroorganism in water to a protein solubilisation temperature 10° C. to150° C. above the fermentation temperature without permitting anysubstantial recovery from the conditioning stage and separating one ormore components of the cell as solids from the protein (optionally afterdecomposition of the protein).

The maximum rate of heating is suitably 0.05° to 20° C. and preferably1° to 10° C. per second and the maximum rate of cooling is suitably0.01° to 20° C. and preferably 1° to 10° C. per second. The averageheating and cooling rates will usually be lower as the rate is afunction of the temperature difference between the materials heated andthe source of heat.

The conditioning effect may be partly or wholly reversed by holding themicroorganisms at a fermentation temperature for an undue period in thecourse of heating to the protein separation stage or permitting contactwith larger quantities of certain ions, especially magnesium ions.

It may be desirable to break down nucleic acids in the heat treatmentstage by a subsequent heat treatment stage and/or for example bytreatment with a nuclease. Such treatment may be desirable in order toreduce the viscosity of an aqueous suspension of microorganism debriswhich contains the desired product(s).

If the desired product is a solid for example a PHA and the protein isnot required, protein is preferably decomposed for example by a proteaseoptionally after partial separation of the PHA from protein by physicalmeans and the desired product separated from the solubilised and/ordecomposed protein. If any of the protein after initial solubilisationbecomes denatured and is thereby converted to a solid or if any of theprotein is not solubilised treatment for example with protease todecompose protein is beneficial. In the case of a PHA separation may beeffected, preferably by filtering or centrifuging after furtherprocessing.

It will be appreciated that if a nuclease is to be used this should bedone before any protease is introduced.

Preferably in the production of PHA the heating stage is sufficient tosolubilise part of the non PHA cell material (NPCM) in the originalcells.

The process normally solubilises at least 25% and preferably at least50%, for example at least 70% of the protein originally present in thecell.

The NPCM will generally comprise nucleic acids, lipiris andphospholipids, peptidoglycan, proteinaceous materials includingglycoproteins and, in some cases lipopolysaccharides and othercarbohydrates. The proteinaceous materials generally form at least 40%by weight of the NPCM.

In the production of PHA at least some of any remaining NPCM componentsof the cell are preferably digested, for example solubilised and/ordecomposed, in one or more stages with a solubilising agent, for examplea surfactant or an oxidising agent. The proteolytic enzyme (protease)may be for example pepsin, trypsin, bromelain, papain, ficin, rennin,chymotrypsin, and bacterial or fungal proteolytic enzymes or mixturesthereof. Suitable enzyme compositions are those commonly employed in"biological" washing powders.

In a digestion stage the solubilising agent may be a proteolytic enzymeand a surfactant.

Suitable surfactants are preferably anionic.

The duration of the heat treatment that is required to effect separationof protein from cells will vary with the temperature employed. Whileheating for at least 5 minutes, and preferably at least 10 minutes, maybe required at temperatures of about 100° C., much shorter periods canbe employed at higher temperatures: for example at 150° C., heatingperiods as short as 20 sec. can be used. Any protein separation stageemploying a surfactant will normally be conducted at temperatures above40° C. in order to effect rapid solubilisation by the surfactant.

Although a wide range of pH conditions can be employed for the heatingstep, the conditions are preferably near neutral, e.g. pH 6-8, tominimise the risk of degradation of the HB polymer.

If an enzyme digestion step is employed, the digestion should beconducted at a temperature below that at which the enzyme is denatured.In many cases the denaturing temperature will be below 65° C. but withsome enzymes the denaturing temperature is higher and so, with suchenzymes, digestion temperatures above 65° C. can be employed. It ispreferred that the digestion temperature is however below 80° C.Suitably the temperature is in the range 50° to 70° C.

Where solubilisation is carried out using both a proteolytic enzymecomposition and a surfactant as the process is preferably performed instages with the surfactant digestion stage performed after the enzymestage or stages because the enzyme composition may be de-activated bythe surfactant, and if it is desired to recycle solubilised NPCM to thefermentation step used to produce the microorganism suspension, thepresence of surfactant in the solubilised portion of the suspension maypreclude such use.

In order to obtain further solubilisation of the NPCM, cellular materialmay be contacted with a phospholipase enzyme to solubilise phospholipidswhich generally account for about 6 to 10% by weight of the NPCM of theoriginal cells.

Enzyme treatments are preferably carried out at a pH within the range6.5 to 9.0 until the requisite degree of treatment has been achieved:this will normally take between 0.2 and 2 hours.

The enzyme digestion may be performed in stages, e.g. an initialtreatment with one enzyme composition followed by one or more treatmentstages wherein the same or a different enzyme composition is employed.

Indeed we have found that in some cases a synergistic effect is obtainedusing an enzyme mixture: thus providing the enzymes do not digest oneanother, in some cases the use of an enzyme mixture results in a higherdegree of solubilisation of NPCM than if the enzymes are used alone orsequentially.

The amount of enzyme, e.g. proteolytic enzyme and/or phospholipaseenzyme, required will depend on the nature and activity of the enzyme:typically the amount of proteolytic enzyme composition will be such asto provide 0.5 to 10, preferably 1 to 6, Anson units (AU) of enzyme per100 g of NPCM in the original cells.

The activity of a proteolytic enzyme may be determined by digestingdenatured haemoglobin with the enzyme for 10 minutes at 25° C. and pH7.5. One Anson unit is the amount of enzyme that, at an initial rate,liberates per minute an amount of TCA soluble product which gives thesame colour with phenol reagent as one milli equivalent of tyrosine. Adetailed description of the analytical method is given in a leaflet AF4issued by Novo Industries. Enzymes such as lysozyme may be used tosolubilise peptidoglycan. The addition of a complexing agent such asethylene diamine tetra-acetic acid to the surfactant may be advantageousin assisting solubilisation of NPCM.

We have found that in some cases where the surfactant treatment isconducted after enzymatic digestion, in particular where the heattreatment prior to enzyme digestion was not particularly severe e.g.where the temperature did not exceed 100° C., an emulsion may be formedon such surfactant treatment from which the solids can only be separatedwith difficulty. The addition of cationic flocculants or electrolytes tosuch emulsions are not particularly effective in assisting thatseparation. However acidification to a pH below 2, or the addition of anabsorbent mineral such as Kieselguhr, can assist separation:acidification may however cause precipitation of some of the NPCMsolubilised by the surfactant.

The insoluble residue remaining after the digestion step will comprisethe PHA polymer together with some residual non-solubilised, NPCM.

In a preferred aspect of the invention, after solubilisation of removalof NPCM following enzymatic and/or surfactant digestion, the PHAcontaining material is treated with hydrogen peroxide. Where the bulk ofthe proteinaceous NPCM has been solubilised by proteolytic enzymes,hydrogen peroxide treatment may effect little or no furthersolubilisation of residual NPCM but may be desirable to removediscoloration of the PHA polymer-containing residue. Hydrogen peroxidetreatment may also be beneficial by enabling the PHA polymer-containingresidue to be more readily separated, e.g. by filtration from theaqueous medium.

In other cases, e.g. where proteolytic enzyme digestion has been used tosolubilise only part of the proteinaceous NPCM, and/or where digestionwith a surfactant has been employed, hydrogen peroxide treatment mayeffect removal of a further proportion of NPCM.

Where the NPCM of the PHA polymer-containing residue comprises lipids,e.g. where no digestion with a phospholipase enzyme has been employed,lipids can be removed by washing the HB polymer-containing residue witha solvent, e.g. methanol, in which the lipids are soluble but the HBpolymer is insoluble. Such a solvent washing step may also be desirableas a deodorising step.

By the above procedures an insoluble residue generally containing atleast 90%, preferably at least 95% and more preferably at least 99% byweight of PHA polymer may be obtained.

In some cases the product from the digestion step can be used as such,for example as a moulding material. Alternatively the HB polymer can beextracted by solvent extraction with a solvent for the HB polymer, e.g.a partially halogenated hydrocarbon such as methylene chloride,chloroform, or 1,2-dichloroethane.

EXAMPLE 1

This illustrates the disruption of a strain of Alcaligenes eutrophus(deposited with National Collections of Industrial and Marine BacteriaLimited (NCIMB), 23 St Machar Drive, Aberdeen AB2 1RY, Scotland, UnitedKingdom under Number NCIMB 40529 on 11 November 1992) by heatshock. Twolevel three variable factorial analysis was used to determine theeffects of Storage temperature, Storage time and Heatshock temperatureon the percentage of total cell protein released. It was found that allthree factors were significant and that there was an interaction betweenstorage temperature and storage time. Rheological tests were alsocarried out to determine the effect of cell storage at differenttemperatures on the conditions of DNA after subsequent heatshock.

Introduction

Cell disruption is an important stage in the separation of intracellularproducts from a bacterial culture. Various methods used includehomogenisation, beadmilling and chemical treatment. Heatshock can beused for partial disruption or as a pre-treatment for some othertechnique. One of the main effects of thermal treatment is the breakdownof the permeability barrier of the cell. This can allow the leakage ofintracellular materials into the suspending medium or the introductionof detergents and lytic enzymes into the cytoplasm to causesolubilisation of the cell. Heatshock can also be used to break geneticmacromolecules such as DNA into smaller soluble components preventingthe formation of a highly viscous gel upon release.

The mechanism of heatshock is dependent on many factors including theheating temperature, the storage conditions, cell condition and whetherthe cell has been grown continuously or in a batch. The extent ofdisruption can be quantitatively measured by the release of solubleprotein into the suspending medium. Much research has been carried outinto the release of intracellular components after thermal treatment[Allwood and Russell, 1968; Watson et al, 1987], this study will attemptto show that the release of components is aided by pre-treatment duringstorage before thermal shocking. Also the condition of DNA afterheatshocking can be influenced by the manner in which the cells arestored.

Materials and Methods

1 Protein Release

Samples of cells of the Alcaligenes eutrophus H16 strain werecentrifuged at 13300 g for 5 minutes before being re-suspended in aglycerol buffer [Uhlenhopp, 1975] to an optical density of approximately10 (540 nm) 5 ml units of cell suspension were added to thin walled testtubes which were used for all tests. A two level three factor experimentwas used with the following parameters, Storage time (1 hour and 6hours), Storage temperature (10° C. and 35° C.) and Heatshocktemperature (50° C. and 90° C.). The temperature of the sample wasmonitored by the immersion of a K-type thermocouple probe connected to aPsion Organiser II hand held computer with an SF10 Datalogger (DigitronInstrumentation). After Heatshocking, the samples were cooled on ice toprevent further damage. Heatshocked cell samples were centrifuged at13300 g for 5 minutes to obtain a debris free supernatant. The insolublematerial remaining following the temperature shock disruption procedureis believed to be substantially composed of a solid polyhydroxyalkanoatepolymer comprising mainly hydroxybutyric acid residues, which is knownto be produced in the cells under the growth conditions used. Thesupernatant was retained for soluble protein analysis by the method ofLowry. Statistical analysis was carried out using Yates algorithms andvariance analysis as given by Box et al [1978].

2 Rheology

The factors used for the rheological work were storage temperature (10°or 35° C.), Storage time (1 or 2 hours and 5 or 6 hours) and incubationtime at 35° C. after heatshock (0 or 35 minutes). Four storage timeswere necessary instead of two to allow the rheological tests which take25 minutes to be carried out. 1 ml of lysing solution (2% Sodium DodecylSulphate, 0.02 M EDTA-Na₂, 0.25N NaOH) was added to 4 ml of heatshockedcell samples. The fluids were mixed by gentle rotation of the test tubeto prevent damage to the sample as a result of high shear. The samplewas analysed using a controlled stress rheometer (Carri-Med, Dorking,Surrey) which subjected the sample to a range of shear stresses between0 and 2 Nm⁻² and measured the resulting shear rates (s⁻¹). The rheogramsproduced give a qualitative indication of the condition of the cellularDNA.

                  TABLE 1                                                         ______________________________________                                        Protein Released                                                              Expressed as a Percentage of the Total Cell Protein                           Storage Heatshock   Stored    Stored Stored                                   Time    Temperature at 1° C.                                                                         at 10° C.                                                                     at 35° C.                         ______________________________________                                        -       -           4.31      9.74   4.17                                     +       -           20.76     27.42  6.05                                     -       +           18.55     22.68  15.18                                    +       +           35.58     38.12  14.13                                    ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                        Calculated Effects and Standard Errors for the Variables Studied                       Levels of Storage Temperature Examined                               Effect     1 and 10° C.                                                                      10 and 35° C.                                                                      1 and 35° C.                         ______________________________________                                        Av. Main Effects                                                                         22.15 ± 0.88                                                                          17.19 ± 0.73                                                                           14.84 ± 0.72                             Storage time :A                                                                          16.65 ± 1.76                                                                          8.49 ± 1.45                                                                            8.58 ± 1.44                              Storage temp :B                                                                          4.69 ± 1.76                                                                           -14.61 ± 1.45                                                                          -9.92 ± 1.44                             Heatshock temp                                                                           13.16 ± 1.76                                                                          10.68 ± 1.45                                                                           12.04 ± 1.44                             :C                                                                            Interactions                                                                  A × B                                                                              -0.09 ± 1.76                                                                          -8.07 ± 1.45                                                                           -8.16 ± 1.44                             A × C                                                                              -0.42 ± 1.76                                                                          -1.30 ± 1.45                                                                           -0.59 ± 1.44                             B × C                                                                              -1.36 ± 1.76                                                                          -1.13 ± 1.45                                                                           -2.49 ± 1.44                             A × B × C                                                                    -0.71 ± 1.76                                                                          -0.17 ± 1.45                                                                           -0.88 ± 1.44                             ______________________________________                                    

Discussion

Table 1 shows evidence that the release of protein from heatshockedcells is much lower when they have been stored at 35° C. than whenstored at 1° C. or 10° C. This could be due to the phospholipids in themembrane being held below their transition temperature forming a rigidstructure (de Mendoza, 1983) which could be ruptured by heatshock moreeasily than a fluid cell membrane. Table 2 gives an indication of thesignificance of factors and whether they interact, As the cells storedat 1° C. and 10° C. give similar results there are no significantinteractions evident but all of the factors are important individually.Comparing cells stored at 1° C. and 35° C. or 10° C. and 35° C. howevershows an interaction between storage time and storage temperature inaddition to the significance of the single factors. This interactioncould be due to the rigid structure remaining but with a slight increasein the content of unsaturated fatty acids due to homeoviscous adaptation(Sinensky, 1974) which would weaken the structure of the wall whenraised to elevated temperatures. The rheograms in contrast to theprotein release indicate that when cells are stored at reducedtemperatures, DNA survives heatshock in a better condition than when thecells have been stored at ambient growth temperatures, we think thatthis could be due to protein binding at low temperatures which protectsvulnerable parts of the DNA molecule when it is heated. A similarphenomenon has been reported in Drosophila which was a 110 kDA proteinwhich binds to nuclease sensitive sections of the DNA molecule whenthere is a change in temperature (Wu 1987). Cells stored at 35° C. showsevere DNA damage which is probably due to strand melting caused whenthe samples were taken above DNA's melting point.

References

Allwood, M. C. Russell, A. D. (1968) Thermally Induced Ribonucleic AcidDegradation and Leakage of Substances from the Metabolic Pool inStaphylococcus aureus. J. Bact. 85(2), 345-349.

Box, G. E. P., Hunter, W. G., Hunter, J. S. (1978) Statistics forExperiments; An Introduction To Design, Data Analysis And ModelBuilding. John Wiley & Sons. pp306-351.

de Mendoza, D. Cronan. J. E. (1983) Thermal Regulation of Membrane LipidFluidity in Bacteria. Trends Biochem. Sci. 8, 45-52.

Sinensky, M. (1974) Homeoviscous Adaptation--A Homeostatic Process thatRegulates the Viscosity of Membrane Lipids in Escherichia coil. Proc.Nat. Acad. Sci. US. 71(2), 522-525.

Uhlenhopp, E. L. Zimm, B. H. (1975) Viscoelastic Characterisation ofSingle-Stranded DNA From Escherichia coli. Biophysical Journal, 15,223-232.

Watson, J. S. Cumming, R. H. Street, G. Tuffnell, J. M. (1978) Releaseof Intracellular Protein by Thermolysis. Separations for Biotechnology.Ed. Verrall, M. S. Hudson, M. J. Ellis Horwood pp 105-109.

Wu, C. et al. (1987) Purification and Properties of Drosophila HeatShock Activator Protein. Science 238, 1247-1253.

EXAMPLE 2

Protein release After Storage at 1° C. and 35° C. Followed by Heatshock

Samples of cells of the Alcaligenes eutrophus H16 strain grown ascontinuous culture were centrifuged at 13400 g for 5 minutes beforebeing resuspended in a glycerol buffer [Uhlenhopp and Zimm, 1975] to anoptical density of 10 (540 mm). 5 ml units of cells were placed in thinwalled test tubes which were stored in water baths at either 1° C. or35° C. for up to 7 hours. Samples were removed every 30 minutes and weretreated by heatshock at 90° C. by immersion in silica oil at 130° C.This was monitored by the immersion of a K-type thermocouple probe intothe sample. This was connected to a Psion II hand held computer with anSF10 Datalogger (Digitron Instrumentation, Hertford, Hertfordshire, UK).After Heatshocking the samples were incubated at 35° C. for 30 minutesin a waterbath before being cooled on ice. Cold samples were immediatelycentrifuged at 13400 g for 5 minutes to obtain a debris free supernatantwhich was stored in a refrigerator before soluble protein analysis bythe method of Lowry. The insoluble material remaining following thetemperature shock disruption procedure is believed to be substantiallycomposed of a solid polyhydroxyalkanoate polymer comprising mainlyhydroxybutyric acid residues, which is known to be produced in the cellsunder the growth conditions used.

References

Uhlenhopp, E. L. Zimm, B. H. (1975) Viscoelastic Characterisation ofSingle-Stranded DNA From Escherichia coli. Biophysical Journal 15,223-232.

Note: It has since been determined that the incubation stage in theprocess will have had no effect on the effect of protein release in thisprocess and was unnecessary as a process step.

We claim:
 1. A process of producing a polyhydroxyalkanoic acid in whicha solid polyhydroxyalkanoic acid is recovered from a solidpolyhydroxyalkanoic acid-producing microorganism of the species Nocardiaor Alcaligenes which comprises growing the microorganism at afermentation temperature, conditioning it to render it susceptible tothermal shock in a conditioning stage by cooling it below thefermentation temperature without any substantial freezing, solubilisingprotein contained in the microorganism by heating the microorganism inwater to a protein solubilisation temperature 10° C. to 150° C. abovethe fermentation temperature without permitting any substantial recoveryfrom the conditioning stage and separating polyhydroxyalkanoic acid as asolid from the protein.
 2. A process according to claim 1 in which thetemperature in the conditioning stage is at least 20° C. below thetemperature of the fermentation stage.
 3. A process according to claim 1in which the conditioning stage is carried out at a temperature in therange 1° C. to 15° C.
 4. A process as claimed claim 1 in which theconditioning stage is continued for 4 to 12 hours.
 5. A processaccording to claim 1 in which the temperature in the protein separationstage is 15° to 120° C. above that of the fermentation stage.
 6. Aprocess as claimed in claim 1 in which the temperature in the proteinsolubilisation stage is 50° to 100° C.
 7. A process as claimed in claim1 in which the protein solubilisation stage is carried out at a pH of 6to
 8. 8. A process as claimed in claim 1 in which the microorganismduring or after the protein solubilisation stage is contacted with ananionic surfactant.
 9. A process as claimed in claim 1 in which thepolyhydroxyalkanoic acid is a polymer of hydroxybutyric acid.
 10. Aprocess as claimed in claim 9 in which the polyhydroxyalkanoic acid is acopolymer of hydroxybutyric acid and hydroxyvaleric acid.
 11. A processas claimed in claim 1 in which the microorganism is of the speciesNocardia.
 12. A process as claimed in claim 1 wherein thepolyhydroxyalkanoic acid is separated after decomposition of theprotein.
 13. A process as claimed in claim 3 wherein the conditioningstage is carried out at a temperature in the range of 1° C. to 10° C.14. A process as claimed in claim 1 in which the microorganism is of theAlcaligenes species.